Comparative evolutionary history of two closely related desert plant, Convolvulus tragacanthoide and Convolvulus gortschakovii (Convolvulaceae) from northwest China

Abstract Desert ecosystems are one of the most fragile ecosystems on Earth. The study of the effects of paleoclimatic and geological changes on genetic diversity, genetic structure, and species differentiation of desert plants is not only helpful in understanding the strategies of adaptation of plants to arid habitats, but can also provide reference for the protection and restoration of vegetation in desert ecosystem. Northwest China is an important part of arid regions in the northern hemisphere. Convolvulus tragacanthoides and Convolvulus gortschakovii are closely related and have similar morphology. Through our field investigation, we found that the annual precipitation of the two species distribution areas is significantly different. Thus, C. tragacanthoides and C. gortschakovii provide an ideal comparative template to investigate the evolutionary processes of closely related species, which have adapted to different niches in response to changes in paleogeography and paleoclimate in northwest China. In this study, we employed phylogeographical approaches (two cpDNA spacers: rpl14–rpl36 and trnT–trnY) and species distribution models to trace the demographic history of C. tragacanthoides and C. gortschakovii, two common subshrubs and small shrubs in northwest China. The results showed the following: (1) Populations of C. tragacanthoides in northwest China were divided into three groups: Tianshan Mountains—Ili Valley, west Yin Mountains—Helan Mountains‐Qinglian Mountains, and Qinling Mountains—east Yin Mountains. There was a strong correlation between the distribution of haplotypes and the floristic subkingdom. The three groups corresponded to the Eurasian forest subkingdom, Asian desert flora subkingdom, and Sino‐Japanese floristic regions, respectively. Thus, environmental differences among different flora may lead to the genetic differentiation of C. tragacanthoides in China. (2) The west Yin Mountains—Helan Mountains‐Qinglian Mountains, and Qinling Mountains—east Yin Mountains were thought to form the ancestral distribution range of C. tragacanthoides. (3) C. tragacanthoides and C. gortschakovii adopted different strategies to cope with the Pleistocene glacial cycle. Convolvulus tragacanthoides contracted to the south during the glacial period and expanded to the north during the interglacial period; and there was no obvious north–south expansion or contraction of C. gortschakovii during the glacial cycle. (4) The interspecific variation of C. tragacanthoides and C. gortschakovii was related to the orogeny in northwest China caused by the uplift of the Tibetan Plateau during Miocene. (5) The 200 mm precipitation line formed the dividing line between the niches occupied by C. tragacanthoides and C. gortschakovii, respectively. In this study, from the perspective of precipitation, the impact of the formation of the summer monsoon limit line on species divergence and speciation is reported, which provides a new perspective for studying the response mechanism of species to the formation of the summer monsoon line, and also provides a clue for predicting how desert plants respond to future environmental changes.


| INTRODUC TI ON
Climate changes have posed major challenges for biodiversity conservation (Heller & Zavaleta, 2009;Meng et al., 2021). To effectively ensure the persistence of biodiversity under the threats of climate change, it is vital to accurately determine its effects on species distribution. The effects of the Pleistocene glacial-interglacial cycle strongly influence the geographical distribution and genetic diversity of global species, having resulted in local species-level extinctions, migrations, and induced allopatric speciation (Willis & Niklas, 2004). For instance, many temperate species distributions contracted at southerly refugia during glacial periods and expanded back into northern regions during interglacial periods in Europe (Hewitt, 2000). Similarly, many desert plants' distributions in northwest China receded to relatively moist refugia during the dry glacial period, subsequently expanding during the relatively moist interglacial period (Gao et al., 2014;Jia & Zhang, 2019;Ma et al., 2012;Su et al., 2016;Zhang et al., 2020). Geological changes since the Miocene have also had important effects on plant distribution. For example, previous phylogeographic studies have indicated that environmental changes caused by the uplift of the Tibetan Plateau have led to the differentiation of desert plant populations in northwest China (Jia & Zhang, 2021;Liu et al., 2006;Meng et al., 2015).
Thus, understanding the impacts of climate change and geological processes on the spatial distribution of plants, especially vulnerable desert plants, can not only provide insight into the history of population dynamics, but can also provide a source of data for the mitigation of future climatic and environmental changes.
Central Asia comprises by majority arid and semi-arid zones. The interaction between topographic structure and climatic conditions forms a more complex landscape pattern in the region. The arid region of northwest China is located in Central Asia. The vegetation of this region is characterized through adaptations to drought resistance and infertility tolerance, which have been advantageous for maintaining and restoring desert vegetative ecosystem. Therefore, phylogeographic patterns and the evolutionary history of species in this region have attracted the attention of an increasing number of biologists. Many studies have shown that climate change and orogeny from the Miocene to Pleistocene have contributed greatly to the patterns of genetic diversity among desert plants (Jia et al., 2016;Jia & Zhang, 2019;Ma et al., 2012). The relatively humid semi-arid region provided refugia for desert plants during the glacial period and further provided a dispersal corridor during the interglacial period, which has had an important impact on the evolution of desert plants and the formation of lineages (Jia & Zhang, 2021;Shi & Zhang, 2015;. Northwest China is a very vast region, including a variety of landforms and flora. Understanding the driving factors and mechanisms of species diversity and persistence in this region is of great significance for biogeography, evolutionary biology, and conservation biology in the global arid area. Convolvulus (Convolvulaceae) is widely distributed among the temperate and subtropical regions (Wood et al., 2015). According to the chloroplast markers matK and rbcL, the Convolvulus species in Eurasia are diffused from the Mediterranean and the Middle East (Mitchell et al., 2016). Eight species are distributed in China.
Convolvulus ammannii and C. lineatus are perennial herbs, whereas C. tragacanthoides, C. fruticosus, and C. gortschakovii are subshrubs or small shrubs. Convolvulus tragacanthoides and C. gortschakovii are mainly distributed in Central Asia, whereas C. fruticosus is mainly distributed in Central and Western Asia (Wood et al., 2015). In addition, C. tragacanthoides and C. gortschakovii are widely distributed throughout northwest China and have similar morphologies, while there was no obvious north-south expansion or contraction of C. gortschakovii during the glacial cycle. (4) The interspecific variation of C. tragacanthoides and C. gortschakovii was related to the orogeny in northwest China caused by the uplift of the Tibetan Plateau during Miocene. (5) The 200 mm precipitation line formed the dividing line between the niches occupied by C. tragacanthoides and C. gortschakovii, respectively.
In this study, from the perspective of precipitation, the impact of the formation of the summer monsoon limit line on species divergence and speciation is reported, which provides a new perspective for studying the response mechanism of species to the formation of the summer monsoon line, and also provides a clue for predicting how desert plants respond to future environmental changes.

K E Y W O R D S
200 mm precipitation line, climate change, desert plants, Phylogeographic structure, population dynamics

Biogeography
C. fruticosus has a narrow distribution in China. Morphologically, the obvious difference between C. tragacanthoides and C. gortschakovii is that the two outer sepals of C. gortschakovii are significantly wider than the three inner sepals, whereas the shape of the inner and outer sepals of C. tragacanthoides is similar (Figure 1; Wu et al., 2004;Wood et al., 2015). According to Jia and Zhang (2021), except for C. ammannii, the evolutionary relationship between C. tragacanthoides and C. gortschakovii is recent. In our field investigation, we found that the distribution of C. ammannii broadly overlapped with that of C. tragacanthoides and C. gortschakovii, but the distribution of C. tragacanthoides and C. gortschakovii barely overlapped. Most populations of C. gortschakovii were mainly distributed in areas with an annual average precipitation of <200 mm, whereas populations of C. tragacanthoides were mainly distributed in areas with an annual average precipitation of >200 mm (Figure 2a), indicating a distribution gradient regulated by precipitation. In addition, compared with C. gortschakovii, C. tragacanthoides is found at much high altitudes, mostly at heights of >1500 m (Figure 2b). The humidification of arid area in northwest China is altitudinal dependent. Under the influence of atmospheric circulation system, precipitation increases with elevation under the action of topographic uplift, which has been proved in the Tianshan Mountains and Qilian Mountains (Yao et al., 2016(Yao et al., , 2018Zhang et al., 2008). Therefore, we assumed that the two species occupy different ecological niches separated by precipitation. Thus, C. tragacanthoides and C. gortschakovii provide an ideal comparative template to investigate the evolutionary processes of closely related species, which have adapted to different niches in response to changes in paleogeography and paleoclimate in northwest China. Jia and Zhang (2021) used two plastid regions (rpl14-rpl36 and trnT-trnY intron) and the nuclear ribosomal internal transcribed spacer (ITS) region to analyze 25 populations of C. gortschakovii from northwest China, employing a phylogeographical approach.
Populations of C. gortschakovii were divided into three groups: the Junggar Basin, the Alatau Mountains-central Tianshan Mountains-Tarim Basin, and the Alxa Desert. The Tianshan Mountains are considered to pose as a substantial geographical barrier to gene flow between the Junggar Basin and the Tarim Basin, while the former is considered the ancestral distribution area of C. gortschakovii.
Further, it has been inferred that the Eurasian forest subkingdom plays an important role in the promotion of genetic diversity of xerophytes in the Asian desert flora subkingdom.
In the present study, we conducted a phylogeographical investigation using two plastid regions (rpl14-rpl36 and trnT-trnY intron) and a species distribution model to trace the demographic history of C. tragacanthoides and C. gortschakovii and to discuss any potential phylogeographical events corresponding to paleoclimatic and paleogeographic changes, which may have affected these species.
Specifically, we aimed to: (1) compare the genetic diversity, genetic structure, and phylogeographic pattern of two related species and; (2) compare the responses of plants adapted to different degrees of drought to paleoclimate change. These data will provide new insight into historical vegetation dynamics and plant species evolution in northwest China.

| Sampling, DNA extraction, amplification, and sequencing
We sampled 164 individuals from 35 populations of C. tragacanthoides, covering almost the entire distribution range of the species in China between June and August 2015. In each population, 2-6 individuals were collected, and fresh leaves were rapidly dried in silica gel. The coordinates of each population were recorded using a global positioning system (GPS) unit (Table 1; Figure 2, Figure S1). All silica gel-dried materials and voucher specimens were stored in state key laboratory of desert and oasis ecology of Xinjiang institute of ecology and geography of Chinese academy of sciences.
Total genomic DNA was extracted from silica geldried leaves using a modified CTAB protocol, as described by Doyle and Doyle (1987). Two primer pairs rpl14-rpl36 using CLUSTAL_W (Thompson et al., 1994), and manually adjusted where necessary.
The data of GPS coordinates and sequence for C. gortschakovii
The program was run for 10,000 iterations in the range 2 ≤ K ≤ 10.
A Bayesian cluster analysis was used to assess the genetic structuring of populations of C. tragacanthoides using STRUCTURE v. 2.3.4 (Pritchard et al., 2000). To obtain stable results, the program was run five times independently, the number of iterations and the burn-in were 20,000 and 120,000, respectively, and the genetic packet number (k) was set from 2 to 10. The DeltaK method was used in the Structure Harvester online tool to determine the optimal number of groups. A molecular variance analysis (AMOVA) was performed by Arlequin v. 3.11 (Excoffier et al., 2005) to assess the level of genetic differentiation between geographic groups and populations.  Table 1.
outgroups might limit the biogeographic analysis; thus, the outgroup sequences were excluded from the analysis.

| Combined analysis of C. tragacanthoides and C. gortschakovii
The level of gene flow between C. tragacanthoides and C. gortschakovii was using DnaSP 6 (Rozas et al., 2017). Genealogical relationships among haplotypes of C. tragacanthoides and C. gortschakovii were described in Network v. 4.6.1.3, followed by the median Joining algorithm (Bandelt et al., 1999). A Bayesian cluster analysis was used to assess the genetic structure of populations of C. tragacanthoides and C. gortschakovii using STRUCTURE v. 2.3.4 software (Pritchard et al., 2000). The parameter settings are the same as those described previously. The divergence time between C. tragacanthoides and C.
gortschakovii was investigated using BEAST v.  (Legoux, 1978;Muller, 1981;Pares Regali et al., 1974a, 1974b. A solanum-like fossilized pollen grain was used to calibrate the roots of the Nicotianoideae and Solanoideae clades of Solanaceae species between 23 and 33.9 Ma (Graham, 2010;Martínez-Hernández & Ramírez-Arriaga, 1999 To identify landscape connectivity, the SDMTOOLBOX toolkit (Brown, 2014) was used to convert the species distribution model into a "dispersal cost" layer, combined with the shared chloroplast haplotypes among each population to calculate the sum of all the lowest cost paths, the final result of which was presented in ARCMAP 10.1 (ESRI).

| Analysis of C. tragacanthoides
After alignment, the sequence lengths of rpl14-rpl36 and trnT-trnY were 1018 bp and 878 bp, respectively. GenBank accession numbers of the rpl14-rpl36 and trnT-trnY sequences in Table S4. The total length of the sequence after ligation was 1896 bp. After sequence analysis of 164 individuals from 35 populations, 22 chloroplast haplotypes were identified, including 12 SNPs and five insertion/deletion sites. Among them, haplotypes H1-H7 were distributed in the western region and H8-H22 were distributed in the eastern region.
In the western region, H3 and H2 were the most common haplotypes and existed in seven and six populations, respectively, while H4 and H5 exist in two populations. In the eastern region, H10 was the most common haplotype and was present in 15 populations.
H11 and H17 were less and were distributed among the five populations; H9, H15, H19 and H22 were distributed in two populations.
The other 11 haplotypes were private haplotypes and only existed in one population, accounting for 50% of the total haplotypes. Among the 35 populations, 13 contained only one haplotype, accounting for 44.44% of the total population ( Figure 2c; Table 1).
There was a large number of populations with only one haplotype, and the geographical distribution pattern of haplotypes resulted in a low average genetic diversity (Hs = 0.328) and a high total genetic diversity (H T = 0.833). In populations with more than three individuals, the highest diversity of haplotypes was observed in population S1 (h = 0.8333), and the highest nucleotide diversity was observed in population G6 (π = 1.499 × 10 −3 ).
The PERMUT analysis showed that the N ST (0.882) value of C.
tragacanthoides was significantly higher than that of the G ST (0.607) value, indicative of significant phylogeographic structure.
SAMOVA showed a sharp increase in F CT values from K = 3 to K = 4. When K ≥ 4, the grouping structure begins to disappear. Thus, the grouping scheme corresponding to K = 3 is as follows: (group 1) populations X1-11, belonging to the Tianshan Mountains and Ili River Valleys   (Table 2).

F I G U R E 3 (a) ΔK value of STRUCTURE analysis of Convolvulus tragacanthoides. (b)
The ΔK value of STRUCTURE analysis of C. tragacanthoides and Convolvulus gortschakoviis. (c) Bayesian assignment probability analysis of C. tragacanthoides. Four inferred groups were represented by four colors (red, green, blue, and yellow). (d) Bayesian assignment probability analysis of C. gortschakoviis and C. tragacanthoides. Three inferred groups were represented by three colors (pink, green, and blue). (e) The network found in C. tragacanthoides and C. gortschakovii. Two red circles indicate missing haplotypes; circle size represents proportional to haplotype frequency.
The MDA analysis was used to estimate population dynamics.
The observed multiple distribution for all samples and the group level rejected a recent history of population expansion ( Figure S2). In contrast, the two test statistics for the selective neutrality of YHQ, QY and Qingling were negative (Table 3)

| Combined analysis of C. tragacanthoides and C. gortschakovii
The total length of the sequence after ligation was 1905 bp. After se-

| Present and past distribution modeling
The MAXENT model was used to simulate the geographical distribution of C. tragacanthoides and C. gortschakovii ( Figure 6, Figure S3).
After 10 cross-validation, the mean values with SD of test AUC were 0.9934 ± 0.0031 and 0.9922 ± 0.004, respectively, indicating that the model had a high goodness of fit to the observation data set. For C.
tragacanthoides, the annual mean temperature (38.3057%) and precipitation of driest month (17.8432%) explained more than half of all variation (56.1489%) observed in the distribution of C. tragacanthoides.
During the LGM, the potential geographic distribution was significantly smaller than that during the present period. For C. gortschakovii, precipitation of coldest quarter (25.4588%), mean temperature coldest quarter (21.0369%), and annual precipitation (12.9671%) explained more than half of the variation (59.46%) in the distribution of the species. Compared with the present geographical distribution range, the potential distribution area in the LGM is smaller. During the mid-Holocene period, the potential geographic distributions of the two species were closer to the present geographic distribution ( Figure S3).
The analysis of the lowest cost path showed that, in the mid-Holocene and at present, C. tragacanthoides had a dispersal corridor along the TI in the western region, and a dispersal corridor along the YHQ and Qinling Mountains in the eastern region, the east and west dispersal corridors are not connected (Figure 7). During the mid-Holocene and that of present times, C. gortschakovii accessed a dispersal corridor connecting its eastern and western regions and a dispersal corridor along the southern part of the Tianshan Mountains (Figure 7).  (Wu, 1979;Wu et al., 2010). The western genetic lineage belongs to the Eurasian forest subkingdom, and the two eastern genetic lineages belong to the Sino-Japanese floristic regions and the Asian desert flora subkingdom. The vegetation types and climates of the three floristic regions were significantly different. Therefore, C. tragacanthoides, which is distributed among different regions, is locally adapted, whereby a similar trend has been observed in other species such as C. gortschakovii (Jia & Zhang, 2021) and Taxus wallichiana (Gao et al., 2007). Qinling Mountains (Wang et al., 2021), and Tianshan Mountains Wang et al., 2010). With the uplift of the mountain, northwest China gradually became more arid. According to the analysis results of the field investigation and SDM, C. tragacanthoides cannot adapt to an arid climate with annual precipitation <200 mm; therefore, post the mountainous uplift events, the groups distributed on the relatively humid hillsides persisted.

Sum of squares
However, populations living in non-uplifted areas exhibited local adaptation or extinction. This result is consistent with those of studies. The climatic and geological changes observed since the Tertiary have had a far-reaching impact on the distribution of flora (Lu et al., 2018;Zhou et al., 2017;Zhu, 2018). Therefore, it is speculated that the genetic differentiation between YHQ, QY, and TI

| Population dynamics of C. tragacanthoides
During the Pleistocene, the periodic cold and drought in the arid area of northwest China had a great impact on the geographical distribution of local plants. The SDM results showed that the present distribution area of the two species was significantly larger than that during the LGM (Figure 6). We also found that the distribution area during the LGM period was significantly reduced and shifted southward of C. tragacanthoides, when compared with the present distribution area (Figure 6). Although MDA rejected a rapid expansion, the haplotype network in the showed a star-like distribution, centered on haplotypes H10 and H11, which was in line with the performance notion of a recent expansion (Figure 3). The neutral test of C. tragacanthoides in the eastern region was also suggestive of a sudden population expansion. Thus, the distribution area of C.
tragacanthoides conformed to the pattern of general species retreating southward during the glacial period and northward during the interglacial period. In the arid area of northwest China, few plants spread from north to south during the interglacial period, and most were affected by the alternation between dry and wet conditions. During the dry glacial period, the plants of this region remained in the humid glacial refugia, and during the humid interglacial period, they dispersed, but there was no obvious north-south diffusion, such as that seen in Atraphaxis frutescens (Xu & Zhang, 2015), C. gortschakovii, Gymnocarpos przewalskii (Jia & Zhang, 2019;Zhang et al., 2020), Clematis sibirica, and C. songorica (Zhang et al., 2013). as Thuja standishii (Worth et al., 2021) and Vincetoxicum japonicum (Li et al., 2016). However, C. gortschakovii originated in the Asian desert flora subkingdom, and like many plants of desert origin, there was no obvious north-south contraction and expansion during glacialinterglacial cycle. The landscape connectivity analysis revealed that the dispersal corridor of C. tragacanthoides was mainly at the edge of TI, YHQ, and QL (Figure 7). This result was consistent with the humidity adaptations exhibited by this species. Previous studies (Jia & Zhang, 2021; have found similar results, having shown that the relatively humid mountain foot in arid area is an important dispersal channel for desert plants.

| Genetic variation in C. gortschakovii and C. tragacanthoides
Interspecific variation among the two species was obvious. The molecular data analysis showed that the two species did not share haplotypes and could be completely separated in the phylogenetic tree and Bayesian cluster analyses (Figures 3, 5). Similarly, according to the SDM analysis, the optimal distribution areas of the two species These results are consistent with those of the field investigation, suggesting that the two species occupy different niches, which may be the cause of interspecies differentiation.
The present study found that the genetic differentiation and geographical isolation zones of the two species in the eastern distribution area were almost consistent with the summer monsoon limit line There are great differences in altitude on both sides of the summer monsoon limit line, and ecological differences caused by complex terrain and climatic factors promote species differentiation (Tang et al., 2018). The formation of the monsoon and nonmonsoon regions is related to the uplift of the Tibetan Plateau and its nearby mountains. Due to the influence of tall mountains, the inland region situated west of the summer monsoon limit line is not significantly affected by the monsoon, with less rainfall and a dry climate, while the climate to the east of the summer monsoon limit line is relatively gortschakovii originated in the west region; thus, the difference in the region of origin may also contribute toward differentiation.
The distribution and evolution of desert vegetation are closely related to many environmental factors, and the precipitation factor is one of the most important factor (Li et al., 2005;Qi et al., 2021;Xu, 2005;Zhang et al., 2017). For example, Li et al. (2005) found that there was a significant positive correlation between normalized vegetation index and precipitation, but the correlation between normalized vegetation index and temperature change was not obvious, indicating that precipitation is the main natural factor affecting vegetation change in northwest China. Similarly, Lu et al. (2018) showed that the mean divergence time of genus of angiosperm assemblages in China was more strongly correlated with annual precipitation than with annual mean temperature. Qian et al. (2020) also found that the diversity and abundance of angiosperms in China is more strongly correlated with annual precipitation than with annual average temperature, and the 500 mm precipitation line is an important dividing line of diversity and abundance. However, there are few reports on how the summer monsoon limit line (the 200 mm precipitation line) affects species distribution. In this study, from the perspective of precipitation, the impact of the formation of the summer monsoon limit line on species divergence and speciation is reported, which provides a new perspective for studying the response mechanism of species to the formation of the summer monsoon limit line and also provides a clue for predicting how desert plants respond to future environmental changes.

ACK N OWLED G M ENTS
We are very grateful to the anonymous reviewers for their help with this manuscript. We would like to thank Editage (www.edita ge.cn) for English language editing. This study was funded by China National Key Basic Research Program (2014CB954201) and Biodiversity Conservation Strategy Program of Chinese Academy of Sciences (ZSSD-012).

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
Sequence data of northwest China Convolvulus tragacanthoide are available on GenBank (http://www.ncbi.nlm.nih.gov/genba nk/) under accession numbers ON338045-ON338066.